The present invention relates to the problem of impurities in preparations of leukocyte interferon which apparently result from the dissociation and reassociation of disulfide bonds during the purification process. Specifically, the invention concerns a novel and effective procedure for removing these impurities from the purified interferon.
Human leukocyte interferon (HuIFN-.alpha.) is representative of leukocyte interferons (IFN-.alpha.)(s) originally produced by cells from vertebrates of various species which share some degree of sequence homology, e.g. bovine leukocyte interferon and leukocyte interferons from canine, piscine or avian species. HuIFN-.alpha. is known to exist in several forms, commonly designated as forms A through K--i.e., for example, HuIFN-.alpha.A and HuIFN-.alpha.D. Some of these have been expressed in E. coli in recoverable quantities as a result of recombinant DNA techniques, and are ordinarily isolated from these cultures by using a monoclonal antibody column as described in Staehlin, et al., J. Biol. Chem., 256: 9750 (1981). It has been found that the interferon so isolated contains contaminants which appear to be products of dissociation and reassociation of disulfide linkages in the native protein. These contaminants are oligomeric forms, which show multiples of the molecular weight of the monomeric protein when subjected to size determination by SDS-PAGE under nonreducing conditions, and also a "slow monomer" which migrates slightly more slowly in SDS-PAGE performed under nonreducing conditions.
One subtype, HuIFN-.alpha.A is believed to contain sulfhydryl groups on amino acids numbers 1, 29, 98, and 138. In the native form, the conformation of the molecule corresponds to linkages of these groups from amino acids 1-98, and from 29-138. It is believed that the 29-138 linkage is required for activity, but that activity will be maintained even if the 1-98 linkage is broken. While it is not intended that the invention be construed to depend on any particular theory of origin for the contaminants, it is currently thought that in the case of HuIFN-.alpha.A, "slow monomer" is derived from disruption of the 1-98 linkage while the 29-138 linkage remains intact, and the set of oligomers, which are less active, or not active at all, depending on size, represent the binding of one molecule of interferon to another through new disulfide linkages. The presence of the oligomers appears to interfere with the activity of the native HuIFN-.alpha.A, the slow monomer is itself active, but may be immunogenic. Therefore, it would be highly desirable to separate the native form from these contaminants.
It has been possible to remove the oligomer from the preparation by gel permeation techniques. However, recovery is not as good as that obtained in the present invention, and, more importantly, the method fails to separate the slow monomer from the native form.
The present invention succeeds in obtaining high yields in isolating the native protein free from both oligomers and slow monomer.